1. Technical Field
The present invention relates to protein transduction and uses thereof. More specifically, the present invention relates to a protein-induced pluripotent stem cell (piPSC) technology that generate high-quality piPS cells within one week with nearly 100% conversion efficiency and its applications thereof. The present invention also relates to a feeder-free piPSC culture condition and a whole dish expansion without colony picking and clonal expansion and their applications thereof.
2. Description of the Related Art
Embryonic stem (ES) cells are stem cells derived from an embryo. With the present state of technology, the creation of a human embryonic stem cell line requires the destruction of a human embryo, raising a controversial ethical issue for human ES cell research. ES cells are distinguished from other types of cells by two distinctive properties: pluripotency and unlimited self-renewal. Under defined conditions, ES cells are capable of renewing themselves indefinitely, allowing ES cells to be employed as useful tools for both research and regenerative medicine, since we can produce limitless ES cells for continued research or clinical use. ES cells are also able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm, include more than 220 cell types in the adult body. Pluripotency distinguishes ES cells from adult stem cells found in adult tissues; while ES cells can generate all cell types in the body, adult stem cells are multipotent and can only produce a limited number of cell types within this tissue type.
ES cell therapies can be used for regenerative medicine and tissue replacement for injury or disease treatments, such as blood, immune-system and different diseases related genetic diseases, cancers, cardio-vascular disease, juvenile diabetes, Parkinson's and Alzheimer's diseases, wound healing, rheumatoid arthritis, baldness, deafness, blindness, amyotrophic lateral-sclerosis, muscular dystrophy and spinal cord injuries. Besides the ethical concerns of stem cell therapy, there is a technical problem of graft-versus-host disease associated with allogeneic stem cell transplantation. Since the ES cells used for regeneration medicine are from human embryos, immuno-rejection will be always a major problem when transplantation of the ES cells is performed to individual patients for disease treatment. Other potential uses of ES cells include investigation of early human development, study of genetic disease, in vitro systems for toxicology testing and drug screening.
The process for culturing ES cells is quite burdensome. Human ES cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish. The inner surface of the culture dish is typically coated with a feeder layer: mouse embryonic skin cells that have been treated so they will not divide, but provide ES cells a sticky surface attaching to. Feeder cells also release nutrients into the culture medium. Researchers have devised ways to grow embryonic stem cells without mouse feeder cells. This is a significant scientific advance because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
The process for ES cell expansion is also quite time consuming. ES cells form colonies. Usually, the ES cell expansion is first to select individual ES cell colonies and then expand the selected colonies. This significantly slows down the ES cell expansion and may cause higher mutational rate if the selected colonies contain genetic mutations.
In 2006, Shinya Yamanaka and colleagues showed that introduction of four transcription genes: Oct4, Sox2, Klf4 and c-Myc using retroviruses into mouse fibroblasts could generate induced pluripotent stem cells (iPS cells), which display pluripotency. This is a revolutionary discovery since for the first time it is shown that somatic cells can be reprogrammed back to ES-like cells. The iPS cells are believed to be identical to natural ES cells in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, the unlimited self-new property and differentiability. However, the full extent of their relation to natural pluripotent stem cells is still being assessed.
It is believed that iPS cell technology is less ethically controversial, since this technology allows for generation of pluripotent stem cells without human embryo. This technology may also enable generation of patient specific ES cell lines that could potentially be used for cell replacement therapies to treat various human diseases and allows the generation of ES cell lines from patients with a variety of genetic diseases and will provide invaluable models to study hose diseases and for drug screening.
In 2007, several groups showed that the iPS cells generated from mouse fibroblasts via four-gene retroviral delivery produced viable chimera. These groups used Nanog for detection of iPS cells, indicating that Nanog is a major determinant of cellular pluripotency. However, c-Myc is oncogenic and 20% of the chimeric mice developed cancer. In a later study, Yamanaka reported iPSCs could be generated without c-Myc. The process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer.
Also in 2007, iPS cells were generated from human somatic cells, representing a milestone for iPS cell technology. However, the viral transfection systems used inserted the genes at random locations in the host's genome and created major concerns for potential therapeutic applications of these iPSCs, because the created cells might be prone to form tumors. To overcome these dangers, an adenovirus was used to transport the requisite four genes into the genomes of skin and liver cells of mice, resulting in cells identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated, although this method has not yet been tested on human cells. Yamanaka and several other labs have since demonstrated reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies.
In May 2009, two reports by Ding's and Kim's labs reported that both mouse and human iPS cells (piPSCs) were generated by direct delivery of four proteins, which were coded by the four reprogramming genes, thus eliminating the need for viruses or genetic modification of human somatic cells. A recent report further showed that iPSCs could be generated using mRNAs to generate mRNA-induced iPS cells (riPSCs). This solved one of the most challenging safety hurdles associated with personalized stem cell-based medicine and enables scientists to make piPS cells without genetically altering them. Because the proteins do eventually degrade, there should be no trace of their existence in the cells by the time they would be used for experiments or therapies, representing a major breakthrough in the iPS cell technology.
Despite intense worldwide research efforts, the current iPS cell technology still suffers FOUR major problems: 1. Inefficiency; 2. Time consumption; 3. Complex and expensive; and 4. Quality problem.
The current iPS cells technology only converts 0.001-1% somatic cells into iPS cells. For gene delivery using virus vectors, the conversion efficiency from somatic cells to iPS cells could reach 0.1-1%, depending on different starting somatic cells. Using embryonic fibroblast as the starting cells, the conversion efficiency reaches ˜1%; however, using adult somatic cells, the conversion efficiency is <0.1%. For gene delivery using non-virus means, this conversion efficiency is <0.1%. The conversion efficiency for human protein-induced iPS cells is extremely low which only reaches 0.001%.
Additionally, the current iPS cell technology, including both gene delivery and protein delivery, usually takes 4-8 weeks to complete reprogramming of human somatic cells into iPS cells and requires many cycles of gene transfection or protein delivery. This makes the current iPS cell technology a complicated and expensive technology. Furthermore, the quality of iPS cells generated by the current iPS cell technology is questionable in terms of resembling human ES cells. These major technical problems block the translation of iPS cells from its human clinical applications.
The main reason that causes these major problems of the current iPS cell technology is due to the technical challenges of gene delivery and protein delivery, which suffer low efficiency and hard to control stoichiometry of the delivered genes with multiple gene/protein delivery. For iPS cell generation, one has to deliver four genes or four proteins simultaneously into nuclei of somatic cells to turn on the intracellular auto-regulatory circuits that initiates the “stem gene expressions” and silences the “somatic gene expressions”, thus somatic cells can be converted into embryonic stem-like cells.
It is common knowledge that the current gene delivery technology, including both virus vector and non-virus approaches, suffers low efficiency problem for four-gene delivery simultaneously. In addition, there is no guaranty that all four genes are delivered inside cells at an equal stoichiometry. In most cases, the four-gene delivery is random, depending on different delivery vehicles used. To solve this problem, an alternative approach has been developed that inserts all four genes into one virus vector with different linkers between each gene. In this case, only one virus vector is required to be delivered into somatic cells for iPS cell generation, enhanced the delivery efficiency. However, even with this approach, a maximum of iPS cell conversion efficiency is achieved only ˜1% for adult somatic cells and could reach <5% for embryonic somatic cells.
In addition to low efficiency of gene delivery, this approach also suffers another problem: time consuming. Once the reprogramming genes have been delivered, they traffic randomly inside cells and only a small fraction of the genes can reach to nuclei. However, only those somatic cells that have the delivery genes located inside nuclei can be reprogrammed to generate iPS cells. This further significantly reduced the iPS cell conversion efficiency, in order to solve this problem, repeated gene transfection has been performed to enhance the random probability of nuclear incorporation of delivered genes, which is time consuming. It takes 7-14 days for the delivered genes to start expressions and ˜30 days to observe non-iPS cell colony formation. To complete reprogramming, it usually takes 4-8 weeks to generate iPS cells from human adult somatic cells.
For protein-induced cell reprogramming, one has to deliver proteins into the nuclei of fibroblasts. Both Ding's and Kim's labs engineered the reprogramming proteins by adding a cell-penetrating peptide (CPP, 9R-11R) into the C-terminus. Although the CPP-fusion method delivers the reprogramming proteins into the cells, several major drawbacks of the CPP-based protein delivery do exist:                (1) The CPP-fusion has a high risk of altering the properties/functions of reprogramming proteins.        (2) The CPP has low protein delivery efficiency.        (3) The CPP-delivered proteins are sensitive to intracellular proteases since CPP is peptide based, causing degradation of the delivered proteins.        (4) The CPP does not have a targeting capability to nuclei for proteins to initiate reprogramming.        
Once the proteins are inside cytosol, the intracellular proteases will first try to degrade them if they are not folded properly. For those proteins that survived intracellular protease degradation, they will randomly collide with different intracellular compartments and only a very small fraction of proteins can reach nuclei to initiate protein-induced reprogramming. To enhance the probability of nuclei location of the reprogramming proteins, both Ding's and Kim's lab adopted a repeated circles (7-10 cycles) of protein delivery.
These major drawbacks cause extremely low efficiency of somatic cell conversion into iPS cells (<0.005%). In addition, it also took a long time for protein initiated reprogramming of fibroblast cells into iPS cells. In Ding's paper, it took 5 weeks to observe iPS cell colony formation from mouse embryonic fibroblast, which is much easy to reprogram into iPS cells as compared with human adult fibroblast. When human newborn fibroblasts were used by Kim et al to generate iPS cells, it took 8 weeks to observe iPS cell colony.
As discussed above, lack of delivery efficiency, lack of nuclear targeting/random nature of intracellular trafficking and long process of the current gene and protein delivery technology make it very hard to control the quality of the generate iPS cells. This is demonstrated by several recent reports, indicating that the newly generated iPS cells display distinct patterns of gene expression from those of human ES cells. However, the iPS cells display a very similar gene expression pattern as human ES cells after 50-60 passages. Based on this results, a continue reprogramming concept has been proposed during continue passage of iPS cells. Unfortunately, it was also observed that at later passages (50-60 passages), the iPS cells display major chromosomal changes as compared with the starting somatic cells, making it impossible to use these IPS cells for human clinical application.
It would therefore be beneficial to develop an iPS cell technology that can generate high-quality iPS cells from human adult somatic cells within a few days at near 100% conversion efficiency.